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32 33 Keywords: segmentation clock, somitogenesis, human mesoderm development, induced 34 pluripotent stem cells, step-wise in vitro mesoderm differentiation, disease modeling 35 Pluripotent stem cells (PSCs) have increasingly been used to model different aspects 36 of embryogenesis and organ formation 1 . Despite recent advances in the in vitro 37 induction of major mesodermal lineages and mesoderm-derived cell types 2,3 , 38 experimental model systems that can recapitulate more complex biological features 39 of human mesoderm development and patterning are largely missing. Here, we 40 utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of 41 presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human 42 somitogenesis. We focused initially on modeling the human segmentation clock, a 43 major biological concept believed to underlie the rhythmic and controlled 44 emergence of somites, which give rise to the segmental pattern of the vertebrate 45 axial skeleton. We succeeded to observe oscillatory expression of core segmentation 46 clock genes, including HES7 and DKK1, and identified novel oscillatory genes in 47 human iPSC-derived PSM. We furthermore determined the period of the human 48 segmentation clock to be around five hours and showed the presence of dynamic 49 traveling wave-like gene expression within in vitro induced human PSM. Utilizing 50 CRISPR/Cas9-based genome editing technology, we then targeted genes, for which 51 mutations in patients with abnormal axial skeletal development such as 52 spondylocostal dysostosis (SCD) (HES7, LFNG and DLL3) or spondylothoracic 53 dysostosis (STD) (MESP2) have been reported. Subsequent analysis of patient-like 54iPSC knock-out lines as well as patient-derived iPSCs together with their genetically 55 corrected isogenic controls revealed gene-specific alterations in oscillation, 56 synchronization or differentiation properties, validating the overall utility of our 57 model system, to recapitulate not only key features of human somitogenesis but also 58 to provide novel insights into diseases associated with the formation and patterning 59 of the human axial skeleton. 60 We initially aimed to mimic and recreate in vitro the signaling events responsible 61for the step-wise emergence of PSM and its derivatives during embryonic development, 62as also recently attempted by others 2,4,5 , via selective activation or inhibition of 63 appropriate signaling pathways, using human iPSCs as starting material (Fig. 1a). We 64 characterized the ability of our in vitro induced human PSM cells to differentiate into 65 somitic mesoderm and its two main derivatives, sclerotome and dermomyotome, which 66give rise to bone and cartilage of the axial skeleton and skeletal muscle and dermis of the 67 emerging embryo respectively. RNA-sequencing (RNA-seq) analysis and subsequent 68 characterization of in vitro derived human PSM samples revealed, that at each step of our 69 induction and differentiation protocol, markers expected to be pr...

Our understanding of human somitogenesis is limited and largely based on insights gained from model organisms. Pluripotent stem cell-based in vitro approaches aiming to recapitulate distinct aspects of this core developmental process have recently been reported, including our recent paper on the in vitro recapitulation of the human segmentation clock 1 . Here we describe in detail our stepwise induction protocol of presomitic mesoderm (PSM), somitic mesoderm (SM), and its two major derivatives, sclerotome (SCL) and dermomyotome (DM) from human induced pluripotent stem cells (iPSCs). We further briefly address the subsequent molecular and functional analysis of these in vitro induced human mesodermal lineages and cell-types.

Our understanding of human somitogenesis is limited and largely based on insights gained from model organisms. Pluripotent stem cell-based in vitro approaches aiming to recapitulate distinct aspects of this core developmental process have recently been reported, including our recent paper on the in vitro recapitulation of the human segmentation clock1. Here we describe in detail our stepwise induction protocol of presomitic mesoderm (PSM), somitic mesoderm (SM), and its two major derivatives, sclerotome (SCL) and dermomyotome (DM) from human induced pluripotent stem cells (iPSCs). We further briefly address the subsequent molecular and functional analysis of these in vitro induced human mesodermal lineages and cell-types.